2-amino-3-aroyl-4,5 alkylthiophenes: agonist allosteric enhancers at human A1 adenosine receptors

ABSTRACT

The present invention relates to a compound of formula (I):                    
     wherein: 
     R 3  is selected from the group consisting of 1-napthyl, 2-napthyl and cycloalkylphenyl; and 
     R 4  and R 5  taken together form a ring having from 5 to 10 carbon atoms. 
     Additionally, the invention provides a therapeutic method for preventing or treating a pathological condition or symptom in a mammal subject, such as a human, wherein increased angiogenesis is desired, comprising administering to a mammal in need of such therapy an effective amount of the aforementioned thiophene selective adenosine A 1  allosteric enhancer.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/292,092 filed in the United States Patent Office on May 18, 2001.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the use of thiophene derivatives asallosteric enhancers of agonist activity at adenosine A₁ receptors.

BACKGROUND OF THE INVENTION

Angiogenesis, the process of new blood vessels formation, is a complexprocess involving the coordinated interaction of numerous cell types.The critical cells are the endothelial cells, which contain all of thegenetic information necessary to form primitive tubes and branches.Other cells, such as smooth muscle cells, mast cells, and macrophagesrelease important modulators of angiogenesis. Hypoxia, decreased bloodflow, and released angiogenic substances such as vascular endothelialgrowth factor (VEGF) can trigger angiogenesis. The process begins with abreakdown of the extracellular matrix, followed by proliferation andmigration of endothelial cells into the tissue. Initially theendothelial cells form cords. Later large vacuoles form in the cells,leading to the formation of tubes. The endothelial tubes have a lumen,but are abnormally permeable and leaky until pericytes are recruited toreinforce the new vessels. Several growth factors, most notably VEGF,bFGF, and angiopoetin-1, promote angiogenesis. VEGF, a specific mitogenfor endothelial cells, can independently stimulate new vessel growth.However, overexpression of VEGF in developing avian embryos results inlarge vessels that are leaky, which leads to tissue edema. Thecoordinated effects of several growth factors may be necessary tostimulate the development of normal new vessels. Hence, finding ways touse upstream modulators in a tissue-specific way may provide atherapeutic advantage over the application of individual growth factors.

VEGF is a direct, or primary, angiogenic factor, meaning that it is ableby itself to induce angiogenesis in endothelial cells in vitro or invivo. Secondary, or indirect, angiogenic factors work by causing cellsto release primary factors. Experts fear that using primary factorsclinically will cause pathologic angiogenesis in other tissues. Thus, alimitation of using adenosine or other promoters of angiogenesis couldbe new vessel growth in healthy as well as diseased tissues. Hence,activation of upstaeam secondary angiogenic stimuli may produce moreregulated and normal vascular growth. Additionally, the ability totarget angiogenic stimulation to specific tissues would diminish therisk of indiscriminate angiogenesis.

There are widespread clinical applications for the stimulation of theangiogenesis in cardiovascular medicine and ophthalmology. Stimulatingnew vasculature in ischemic tissues, especially heart and limbs iscurrently an active clinical endeavor because it could have a majorimpact on morbidity and mortality from atherosclerosis. Trials in humanshave shown the usefulness of VEGF in stimulating collateral vessels toischemic lower extremities, improving ulcer healing and decreasing limbloss. There are also ongoing clinical trials using VEGF infusions inpatients with intractable, inoperable angina pectoris.

Abundant evidence shows that hypoxic or ischemic tissues releaseadenosine and that adenosine stimulates angiogenesis. Possiblemechanisms of vessel growth include increased flow, stimulation ofvascular cell proliferation and migration, or stimulation of growthfactor secretion. Some of the results obtained in previous studies onadenosine effects in vivo and in vitro have suggested that activation ofadenosine A₂ receptors (A_(2A) or A_(2B)) are responsible for theability of adenosine to stimulate angiogenesis. The activation of A_(2B)receptors on cultured endothelial cells has been shown to stimulate VEGFrelease, but A₁ adenosine receptor activation seemed to play little orno role. The present invention, however, demonstrates the A₁ receptor ismore important than has been previously thought. Indeed, the presentinvention relates to the use of thiophene derivatives as allostericenhancers of agonist activity at adenosine A₁ receptors. Allostericenhancers of A₁ adenosine receptors selectively stimulate angiogenesisin ischemic tissue and not in tissue that has adequate blood flow. Thissite-specificity represents a major advantage over other angiogeneicagents that are not selective for ischemic tissue.

Adenosine triggers endothelial cell proliferation in cultured cells andangiogenesis in animal models. Adenosine is a logical modulator for thehypoxic stimulation of angiogenesis. It is a metabolite of ATP releasedfrom all ischemic or hypoxic tissues, where it acts as a “retaliatorymetabolite” to restore normal oxygen delivery, initially by dilatingexisting blood vessels. Chronic hypoxia has long been considered adriving force for new blood vessel formation. Increased vascular densityis seen in humans at high altitudes, in chronically stimulated skeletalmuscle, and in rapidly growing tumors. Hypoxia initiates proliferationof cultured endothelial cells that can be blocked by unselectiveadenosine receptor antagonists. Subtype-selective ligands have been usedto tease out the mechanism of adenosine-induced endothelial cellproliferation and migration, but the results have been inconsistent. Thechicken chorioallantoic membrane (CAM) model is a suitable vehicle forstudying the effect of adenosine on angiogenesis. In this model loweringoxygen concentration stimulates neovascularization, but adenosine hasnot been consistently angiogenic. Receptor subtype-selective ligandshave not previously been tested in the CAM.

Adenosine acts via four types of cell surface, G protein-coupledreceptors, A₁, A_(2A), A_(2B) and A₃. A₁ and A₃ receptors are themostsimilar in amino acid sequence and pharmacology. These receptors coupleto G proteins from the Gi/Go family and inhibit adenylyl cyclase.Stimulation of A₁ and A₃ receptors can also activate phospholipase C,presumably via G protein sub-units. A_(2A) and A_(2B) receptors coupleto Gs and stimulate adenylyl cyclase, but the A_(2B) receptor can alsocouple to Gq. In the heart, A₁ receptors have negative chronotropic,dromotropic and inotropic effects. The A₁ receptor, and perhaps the A₃,is also involved in the preconditioning phenomenon, which protectsischemic tissues. Coronary arteries express A_(2A) receptors; theiractivation results in coronary vasodilation. A_(2A) receptors also occuron leukocytes, where they attenuate the inflammatory response andthereby decrease reperfusion injury. Accordingly, adenosine acts in anumber of ways to protect ischemic tissues; it decreases metabolism,increases blood flow, and attenuates inflamnmatory injury. Adenosineactivates A_(2B) receptors on cultured endothelial cells to trigger VEGFrelease and endothelial mitogenesis. Adenosine also appears to stimulateangiogenesis, but to date no attempt has been made to define theadenosine receptor subtypes involved in the CAM model. Additionally,heretofore, it had not been shown that adenosine stimulates angiogenesisin adult mammalian models. The development of more selective adenosinereceptor ligands and cloning of the chicken A₁, A_(2A), and A₃ receptorshave enabled us to identify adenosine receptor subtypes participating inthe angiogenic response of CAM.

Allosteric enhancers of receptors are defined as compounds that bind toan allosteric site distinct from the binding site of the endogenousligand and potentiate responses to agonists. Benzodiazepine anxiolyticsand calcium channel blockers are familiar examples of drugs that actallosterically. Allosteric enhancers of adenosine A₁ receptors act onlyon the adenosine-receptor-G protein ternary complex. Accordingly, theyhave little effect by themselves, but enhance the actions initiated byA₁ receptors when increases in endogenous adenosine levels in ischemictissues increase receptor occupancy.

PD 81,723 (PD) is the archetype of a family of aminothiophenes that werethe first described allosteric enhancers of adenosine A₁ receptors.These compounds increase binding of [³H]N⁶-cyclohexyladenosine (CHA) toadenosine A₁ receptors and caused a functional enhancement of theeffects of adenosine A₁ receptor activation in various tissues. PD isselective for adenosine A₁ receptors, having no effects on otheradenosine receptor subtypes or on other classes of receptors. PD hasshown enhancement at A₁ receptors of all species tested to date. In theabsence of adenosine or A₁-selective agonists, the enhancer moleculesalone act as very weak antagonists for adenosine receptors. Despite PDdemonstrating allosteric enhancer activity, there still remains a needfor compounds having improved allosteric enhancer activity.

The administration a compound that promotes angiogenesis can be aneffective method for treating stroke, heart disease, peripheral vasculardisease. The administration of such compound can also be an effectivemethod for treating cardiac arrhythmias, chronic pain and inducingsleep. The ability of the improved allosteric enhancers described hereinto promote angiogenesis in two animal model systems, the chickenchorioallantoic membrane model and the rat mesenteric model,demonstrates that allosteric enhancers of the adenosine A₁ receptorenhance the ability of adenosine to promote new vessel growth.

Accordingly, the present invention provides novel thiophene derivativesto be used as improved agonist allosteric enhancers at adenosine A₁receptor.

Additionally, the present invention provides an original therapeuticmethod for preventing or treating a pathological condition or symptom ina mammalian subject, such as a human, wherein increased angiogenesis isdesired, by administering to a mammal in need of such therapy aneffective amount of the aforementioned adenosine A₁ receptor allostericenhancer.

SUMMARY OF THE INVENTION

One aspect of the present invention is a compound of Formula I:

wherein R₃ is selected from the group consisting of 1-napthyl, 2-napthyland cycloalkyl-phenyl; and

R₄ and R₅ are taken together to form a ring having 5 to 10 carbon atoms.

A second aspect of the present invention is a method of allostericallyenhancing adenosine A₁ receptors in a mammal, including a human, by theadministration to the mammal of an amount of a compound of Formula Isufficient to enhance actions mediated by adenosine receptors.

A third aspect is a pharmaceutical formulation comprising a compound ofFormula I and one or more excipients.

A fourth aspect is a method of treating ischemic disease in a mammal,including a human, by administering an effective amount of a compound ofFormula I.

BRIEF DESCRIPTION OF THE DRAWING

FIGURE one is a schematic demonstrating the synthesis of2-amino-3-benzoyl-4,5-dimethylthiophenes (compounds 4-17).

DETAILED DESCRIPTION

Specific compounds of the present invention are (Formula I):

wherein:

R₃ is selected from the group consisting of 1-napthyl, 2-napthyl and1-cycloalkyphenyl; and 1-napthyl and 2-napthyl are optionallysubstituted with (C₁-C₆)alkyl groups, (C₂-C₆)alkenyl groups,(C₁-C₆)alkanoyl groups, (C₁-C₆)alkanoyloxy groups, (C₃-C₆) cycloalkylgroups, (C₃-C₆) cycloalkenyl groups, halo (C₁-C₆)alkyl groups,(C₁-C₆)alkoxy groups, (C₁-C₆)alkoxycarbonyl groups,(C₃-C₆)cycloalkyl(C₁-C₆)alkyl groups, (C₂-C₆)alkynyl groups, cyano orone of more halogen atoms, such as fluorine, chlorine, bromine oriodine; and

R₄ and R₅ are taken together to form an unsaturated or saturated ringhaving from 5 to 10 atoms.

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc.denote both straight and branched groups; but reference to an individualradical such as “propyl” embraces only the straight chain radical, abranched chain isomer such as “isopropyl” being specifically referredto.

Specific and preferred values listed below for radicals, substituents,and ranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents.

Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;(C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl; (C₃-C₆)Cycloalkenyl can be cyclopropenyl, cyclobutenyl,cyclopentenyl, or cyclohexenyl; (C₃-C₆)cycloalkyl(C₁-C₆)alkyl can becyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl,2-cyclopentylethyl, or 2-cyclohexylethyl; (C₁-C₆)alkoxy can be methoxy,ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, pentoxy,3-pentoxy, or hexyloxy; (C₂-C₆)alkenyl can be vinyl, allyl, 1-propenyl,2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl,3-pentenyl, 4pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or5-hexenyl; (C₂-C₆)alkynyl can be ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl,4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl;(C₁-C₆)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C₁-C₆)alkylcan be iodomethyl, bromomethyl, chloromethyl, fluoromethyl,trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, orpentafluoroethyl; (C₁-C₆)alkoxycarbonyl can be methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, or hexyloxycarbonyl; (C₁-C₆)alkanoyloxy can be acetoxy,propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy.The preceding examples are illustrative, not exhaustive.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. It is to be understood that thepresent invention encompasses any racemic, optically-active, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine agonist or antagonist activityusing the standard tests described herein, or using other similar testswhich are well known in the art.

Processes for preparing compounds of Formula I or for preparingintermediates useful for preparing compounds of Formula I are providedas further embodiments of the invention. Intermediates useful, forpreparing compounds of Formula I are also provided as furtherembodiments of the invention.

In cases where compounds are sufficiently basic or acidic to form acidor base salts, use of the compounds as salts may be appropriate.Examples of acceptable salts are organic acid addition salts formed withacids that form a physiological acceptable anion, for example, tosylate,methanesulfonate, acetate, citrate, malonate, tartarate, succinate,benzoate, ascorbate, alpha-ketoglutarate, and alpha-glycerophosphate.Suitable inorganic salts may also be formed, including hydrochloride,sulfate, nitrate, bicarbonate, and carbonate salts.

Acceptable salts may be obtained using standard procedures well known inthe art, for example by reacting a sufficiently basic compound such asan amine with a suitable acid affording a physiologically acceptableanion. Alkali metal (for example, sodium, potassium or lithium) oralkaline earth metal (for example calcium) salts of carboxylic acids canalso be made.

The ability of a compound of the invention to enhance the affects ofadenosine may be determined using pharmacological models well known tothe art, or using the assays described herein below.

Compounds of this invention may be useful for: (1) protection againsthypoxia- and/or ischemia-induced injuries (e.g., stroke, infarction);(2) treatment of adenosine-sensitive cardiac arrhythmias; (3)antinociception (i.e., analgesics); (4) anticonvulsants; (5) sleepinduction, (6) treatment of chronic pain and (5) other indications forwhich A₁ agonists are used.

The amount of compound of the present invention required to be effectiveas an allosteric enhancer of an adenosine receptor will, of course, varywith the individual mammal being treated and is ultimately at thediscretion of the medical or veterinary practitioner. The factors to beconsidered include the condition being treated, the route ofadministration, the nature of the formulation, the mammal's body weight,surface area, age and general condition, and the particular compound tobe administered.

Formulations of the present invention for medical use comprise an activecompound, i.e., a compound of Formula (I) with a pharmaceuticallyacceptable carrier thereof and optionally other therapeutically activeingredients. The carrier must be pharmaceutically acceptable in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof.

The present invention, therefore, further provides a pharmaceuticalformulation comprising a compound of Formula (I) with a pharmaceuticallyacceptable carrier thereof.

The formulations include, but are not limited to, those suitable fororal, rectal, topical or parenteral (including subcutaneous,intramuscular and intravenous) administration. Preferred are thosesuitable for oral or parenteral administration.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active compound intoassociation with a carrier that constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier or a finely divided solid carrier and then, if necessary,shaping the product into desired unit dosage form.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the active compound;as a powder or granules; or a suspension or solution in an aqueousliquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or adraught.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface activeor dispersing agents. Molded tablets may be made by molding a mixture ofthe powdered active compound with any suitable carrier in a suitablemachine.

A syrup or suspension may be made by adding the active compound to aconcentrated, aqueous solution of a sugar, e.g., sucrose, to which mayalso be added any accessory ingredients. Such accessory ingredients mayinclude flavoring, an agent to retard crystallization of the sugar or anagent to increase the solubility of any other ingredient, e.g., as apolyhydric alcohol, for example, glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppositorywith a conventional carrier, e.g., cocoa butter or Witepsol S55(trademark of Dynamite Nobel Chemical, Germany), for a suppository base.

Formulations suitable for parenteral administration convenientlycomprise sterile aqueous preparation of the active compound, which ispreferably isotonic with the blood of the recipient. Thus, suchformulations may conveniently contain distilled water, 5% dextrose indistilled water or saline. Useful formulations also compriseconcentrated solutions or solids containing the compound of Formula (I),which give a solution suitable for parental administration upon dilutionwith an appropriate solvent.

Topical formulations include ointments, creams, gels and lotions, whichmay be prepared by conventional methods known in the art of pharmacy. Inaddition to the ointment, cream gel, or lotion base and the activeingredient, such topical formulation may also contain preservatives,perfumes, and additional active pharmaceutical agents.

In addition to the aforementioned ingredients, the formulations of thisinvention may further include one or more optional accessoryingredient(s) utilized in the art of pharmaceutical formulations, e.g.,diluents, buffers, flavoring agents, binders, surface active agents,thickeners, lubricants, suspending agents, preservatives (includingantioxidants) and the like.

EXAMPLES

The symbols and conventions used in these examples are intended to beconsistent with those used in the contemporary, international, chemicalliterature, for example, the Journal of the American Chemical Societyand Tetrahedron.

The allosteric enhancer (AE) activity was studied at the human A₁AR(hA₁AR) of a panel of compounds consisting of nine2-amino-3-aroylthiophenes, (3a-i), eight2-amino-3-benzoyl-4,5-dimethylthiophenes (13a-h), three3-aroyl-2-carboxy-4,5-dimethylthiophenes, (17a-e), ten2-amino-3-benzoyl-5,6-ihydro-4H-cyclopenta[b]thiophenes, (19a-I),fourteen 2-amino-3-benzoyl-4,5,6,7-tetrahyd robenzo-[b]thiophenes,(20a-n), and fifteen2-amino-3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes,(21a-o). An in vitro assay employing the A₁AR agonist¹²⁵I-N⁶-aminobenzyladenosine (¹²⁵I-ABA) and membranes from CHO-K1 cellsstably expressing the hA₁AR measured, as an index of AE activity, theability of a candidate AE to slow the dissociation of the radioligandfrom the A₁AR-G protein ternary complex.

Compounds 3a-i had little or no AE activity and compounds 13a-h had onlymodest activity, evidence that AE activity depended absolutely on thepresence of at least a methyl group at C-4 and C-5. Compounds 17a-clacked AE activity, suggesting the 2-amino group is essential.Polymethylene bridges linked thiophene C-4 and C-5 of compounds 19a-j,20a-m and 21a-o. AE activity increased with the size of the —(CH₂)_(n)—bridge, n=3<n=4<n=5. The 3-carbethoxy substituents of 19a, 20a and 21adid not support AE activity, but a 3-aroyl group did. Surprisingly,3-napthoyl and 3-cycloalkylphenyl groups had the greatest enhancingactivity. Particularly, bulky (or hydrophobic) substituents at the metaand para positions of the 3-phenyl group and also 3-naphthoyl groupsgreatly enhanced activity. Thus, the hA₁AR contains an allostericbinding site able to accommodate 3-aroyl substituents that are bulkyand/or hydrophobic but not necessarily planar. A second region in theA₁AR interacts constructively with alkyl substituents at thiophene C-4and/or C-5.

Chemistry,

The reaction of 2,5-dihydroxy-1,4-dithiane (thioacetaldehyde dimer) witharoylacetonitriles gave 2-amino-3-aroylthiophenes 3a-h.

The base-catalyzed condensation of an aryl β-ketonitrile with 2-butanoneto form a mixture of the E- and Z-isomers of2-benzoyl-3-ethylcrotonitrile, followed by cyclization with sulfur is ageneral method for the synthesis of2-amino-3-aroyl-4,5-dimethylthiophenes. However, because only theE-isomer can react with sulfur, low yields are an inherent, disadvantageof that approach. A more efficient alternative synthesis was thereforedeveloped proceeding from 3-methyl-2-thiophenecarboxaldehyde, 4, togenerate 2-amino-3-benzoyl-4,5-dimethylthiophenes 13a-h (Scheme 1). TheHuang-Minlon modification of the Wolf-Kishner reduction of 4 generated2,3dimethylthiophene, 5. Originally, an amino function was to beintroduced at C-2 by nitration of 5 and then reduction of the4,5-dimethyl-2-nitrothiophene. Unfortunately, the nitrothiophene, 6,proved difficult to purify, and the subsequent reduction gave a tar. Theinstability of 2-amino-4,5-dialkylthiophenes is well known. Thealternative synthesis consisted of the tin (IV) chloride-catalyzedFriedel-Crafts acylation of 5 with acetyl chloride to yield2-acetyl-4,5-dimethylthiophene, 7. Forming the oxime, 8, andPCl₅-catalyzed Beckmann rearrangement of that oxime gave a mixture of2-acetamido-4,5-dimethylthiophene, 9, a key intermediate for thesynthesis of 12a-g, as well as N-methyl2-carboxamido-4,5-dimethylthiophene, 10. Friedel-Crafts acylation of 9by benzoyl chlorides 11a-h gave 2-acetamido-3-benzoylthiophenes 12a-h.Solvent importantly affected yield; in the case of acylation withbenzoyl chloride, replacing benzene with 1,2-dichloroethane improvedyield from 47% to 82%. Baseatalyzed deprotection of 12a-h gave thetarget thiophenes, 13a-h. Deprotection with acid catalyzed the formationof the dimers such as 14, which lacked AE activity. The dimerization wasnot readily apparent in NMR spectra, but was evident in high-resolutionmass spectrometry. Deprotection of the 2,4,6-trimethylbenzoyl compound12h with acid did not lead to dimerization, perhaps a result of theelectronic or steric effects of the three methyl groups.

Compound 10 is a side product in the pathway leading to 13a-h, but itsderivatives offered the chance to test whether the 2-amino group isimportant for activity. The 2-amino group was replaced with a carboxylgroup, prepared by the hydrolysis of the amide group of 10. Thatapproach failed because the electron-withdrawing effect of the2-substituent made the thiophene resistant to Fridel-Crafts acylation atC-3. However, another approach circumvented that problem. Lithiation of10 to lithiothiophene 15 permitted acylation with benzoyl chlorides,forming the amides, 16a-c. Alkaline hydrolysis then gave compounds17a-c.

The method of Gewald served for the syntheses of5,6-dihydro-4H-cyclopenta[b]thiophenes 19a-h,4,5,6,7-tetrahydrobenzo[b]thiophenes 20a-n and5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes 21a-o. That methodconsists of the base-catalyzed condensation (Knoevenagel) of acycloalkanone 18a-c with an aryl β-ketonitrile to form an olefin.Subsequently, that olefin undergoes cyclization with sulfur to form a2-amino-3-aroylthiophene. Most of the present syntheses followed the“one pot” variant, which consists of adding all the reactants andcatalyst at once, thereby avoiding the necessity of isolating the olefinintermediate before the subsequent reaction with sulfur. The two-stepvariant served for making multigran quantities of 201 and 20n.Diethylamine was usually the catalyst; however, a solid phase catalystgave results similar to those using diethylamine. Neither LiCl, norzeolites, which suffice for Knoevenagel condensations of aldehydes,catalyzed the condensation of ketones.

Bromoacetylarenes were the starting materials for the preparation of theβ-ketonitriles used to synthesize the thiophenes. Since only a few werecommercially available, they were prepared them by reacting acetoareneswith elemental bromine in glacial acetic acid, 1,4-dioxane dibromide,copper (II) bromide or tetrabutylammonium tribromide. Brominations bymeans of Cu(II)Br or tetrabutylammonium tribromide were rapid, clean andnearly quantitative. By contrast, brominations with either Br₂/aceticacid or dioxane dibromide required over two equivalents of brominatingagent to drive the reaction to completion. Reacting thebromoacetylarenes with NaCN in cooled ethanol-water generated theβ-ketonitriles.

Experimental Section.

Melting points are uncorrected. Elemental analyses agreed within ±0.4%of calculated composition. ¹H NMR spectra were consistent with theputative structures. Trans-World Chemicals, Rockville, Md., supplied3′-iodoacetophenone. One recrystallization from methanol removed minorimpurities from 4-acetylbiphenyl. All other starting materials were fromAldrich and were used as received. The brorinations of acetylarenes andtheir conversions to aroylacetonitriles followed the methods cited.

2-Amino-3-benzoylthiophene (3a). A mixture of benzoylacetonitrile (1.45g, 10 mmole), 2,5-dihydroxy-1,4-dithiane (0.76 g, 5 mmole) anddiethylamine (0.73 g=1.04 mL, 10 mmole) in 4 mL absolute ethanol washeated in a teflon-sealed pressure tube for 4 hours at 50° C. withfrequent stirring on a vortex mixer. By 2 hours starting materials haddissolved and shortly thereafter product began to crystallize. Afterrefrigerating the tube overnight, the product was filtered off andwashed with a little methanol to give bright yellow crystals. Yield 1.3g, 64% ¹H NMR (CDCl₃) δ: 6.14 (d, 1H, H-5), 6.88 (d, 1H, H-4), 6.95 (brs, 2H, NH₂), 7.5-7.7 (m, 5H, C₆H₅).

2,3-dimethylthiophene (5). Heating a mixture of 3-methyl-2-thiophenecarboxaldehyde, 4 (58.6 g, 464 mmole), 80% hydrazine hydrate (97 mL,1.62 mole) and 200 mL ethylene glycol to an internal temperature of130-160° C. caused hydrazine and water to distil. The reaction mixturewas cooled to below 60° C. and the water-immiscible fraction of thedistillate was returned to the flask. The addition of KOH (91.0 g, 1.62mole) and reheating caused vigorous gas evolution when the temperaturereached 90-100° C. Reflux continued for 15 minutes after gas evolutionceased; steam distillation then separated 5. Product in the distillatewas extracted into ether, the extract washed with 6 N HCl, dried overCaC₁₂ and evaporated. Distillation over sodium gave 5 as a colorlessoil, bp 139.5-140.5, yield 39.8 g, 77%. ¹H NMR (CDCl₃) δ: 2.21, s, 3HCH₃; 2.41, s, 3H, CH₃; 6.84, d, J=5.1 Hz, 1H, H-4, 7.03, d, J=5.2 Hz,1H, H-5. ¹³C NMR(CDCl₃) δ: 13.0, 13.6, 120.6, 129.9, 132.6, 133.0.

(4,5-dimethyl-2-thienyl)(methyl) methanone (7). A solution of 5 (15.16g, 135 mmole) and acetyl chloride (9.6 mL, 135 mmole) in 60 mL benzenedried over Na was cooled to −5° C. and vigorously stirred during theaddition of a solution of tin (IV) chloride in 50 mL benzene over aperiod of 1 hour. The reaction mixture was removed from the cold bathand stirred for an additional hour at room temperature. The slowaddition of 4 mL concentrated HCl in 28 mL water quenched the reaction.The organic layer was separated, washed with 2×10 mL water, dried overNa₂SO₄ and evaporated to give 20.8 g of crude product. Chromatography ona column of silica eluted with pet. ethenethyl acetate (10:1) andevaporation of relevant fractions gave a viscous yellow oil, 16.42 g,79%. ¹H NMR (CDCl₃) δ: 2.08, s, 3H, COCH₃; 2.31, s, 3H, CH₃; 2.41, s,3H, CH₃; 7.33, s, 1H, H-3. ¹³C NMR (CDCl₃) δ: 13.3, 13.7, 26.1, 134.7135.3, 139.1, 143.4, 190.0.

1-(4,5-dimethyl-2-thiophen-2-yl)-ethanone oxime (8). A mixture of 7(33.1 g, 215 mmole), hydroxylamine hydrochloride (32.9 g, 473 mmole) andbarium carbonate (91.7 g, 495 mmole) in 500 mL ethanol was heated atreflux for 8 hours, the salts filtered and the filtrate evaporated to anoff-white solid. Crystallization from ethanol-water afforded 30.8 g(85%) of pure 8. Four recrystallizations improved the E:Z ratio ofisomers from 4:1 to 14:1. 2.11 (s, 3H, CH₃C═NOH), 2.26 (s, 3H, CH₃),2.32 (s, 3H, CH₃), 6.94 (s, 1H, H-3), 9.62 (br s, 1H, OH. ¹³C NMR(CDCl₃) δ: 11.4, 12.6, 12.9, 129.2, 132.7, 133.1, 135.8, 149.4.

N-(4,5-dimethyl-thiophen-2-yl) acetamide (9) and4,-dimethyl-thiophene-2-carboxylic acid methylamide (10). A solution of8 (0.304 g, 1.8 mmole) in 5 mL dry ether was cooled to 0° C. and stirredvigorously during the addition of PCl₅ (0.4 g, 1.9 mmole) at a rate thatkept the temperature at 0° C. Stirring on ice continued for 15 min andat room temperature for an additional 30 min. The addition of 1 mL waterat a rate keeping the temperature<20° C. quenched the reaction. Undercooling NaOH was added to bring the pH to 5-6, and product was extractedinto ether. Evaporation gave 0.324 g of crude product that was purifiedby elution from a silica gel column with pet ether-ethyl acetate 1:1 togive 9 (0.097 g, 32%). ¹H NMR (CDCl₃) δ: 2.03 (s, 3H, COCH₃), 2.16 (s,3H, CH₃), 2.24 (s, 3H, CH₃), 6.39 (s, 1H, H-3), 9.08 (s, 1H, NHC═O). ¹³CNMR (CDCl₃) δ: 12.3, 13.4, 23.0, 115.6, 124.8, 129.5, 134.2, 167.3.Additional fractions contained 10 (0.060 g, 20%). ¹H NMR (CDCl₃) δ: 2.10(s, 3H, C_(H)3), 2.33 (s, 3H, CH₃), 2.94 (d, 3H, NHCH₃), 6.21 (br s, 1H,NH, 7.22 (1H, ArH. ¹³C NMR (CDCl₃) δ: 13.4, 13.5, 26.6, 131.0, 133.2,134.0, 138.2, 162.8.

N-(3-benzoyl-4,5-dimethyl-thiophen-2-yl)acetakmide (12a). General MethodA. A solution of 1.71 M tin (IV) chloride (3.1 mL, 5.3 mmole) in1,2-dichloroethane was added dropwise to a suspension of 9 (0.241 g,1.42 mmole) and benzoyl chloride (0.31 mL, 2.66 mmole) in1,2-dichloroethane and the mixture was refluxed for 10.5 hours. Thereaction was quenched with ice and the organic phase was washedsequentially with 2N HCl, water and 2N NaOH. Drying over CaCl₂ andevaporation gave a solid that was purified by chromatography on silicagel eluted with pet. ether-ethyl acetate 5:1. Recrystallization fromwater gave 0.32 g of pure product as yellow crystals, 82%. ¹H NMR(CDCl₃) δ: 1.6 (s, 3H, CH₃), 2.23 (s, 3H, CH₃), 2.24 (s, 3H, CH₃),7.4-7.6 (m, 5H, C₆H₅), 11.1 (br s, 1H, NH₂), ¹³C NMR (CDCl₃) δ: 12.4,14.8, 23.6, 122.4, 124.7, 127.6, 128.3, 128.4, 131.9, 140.3, 146.4,167.4, 195.0.

(2-amino-4,5-dimethyl-thiophen-3-yl)(phenyl)methanone (13a). GeneralMethod B. A solution of 12a (0.3 g, 1.1 mmole) in KOH (3.5 equivalentsin methanol-water 1:1) was refluxed for 45 minutes, evaporated and takenup in dichloromethane. The solution was washed three times with water,dried and evaporated to a solid that was recrystallized fromethanol-water as yellow crystals. Yield 0.25 g, 100%. ¹H NMR (CDCl₃) δ:1.5 (s, 3H, CH₃), 2.1 (s, 3H, CH₃), 6.4 (br s, 2H, NH₂), 7.2-7.5 (m, 5H,C₆H₅). ³C NMR (CDCl₃) δ: 12.5, 15.2, 114.9, 117.2, 127.8, 128.0, 128.8,130.4, 141.7, 162.8, 193.0.

4,9-bis-(3-fluorophenyl)-2,3,7,8-tetramethyl-1,6-dithia-5,10-diaza-dicyclopenta[a,e]-cyclooctene(14). A solution of 12a (0.54 g, 1.86 mmole) in ethanolic 0.5 N HCl washeated at reflux for 7 hours, cooled and alkalinized with NaOH.Extracting into dichloromethane, drying and evaporation gave a solidthat was purified by chromatography on silica gel eluted with pet.ether-ethyl acetate 10:1. Crystallization from ethanol-water gave orangecrystals, 0.254 g, 57%. ¹H NMR (CDCl₃) δ: 1.6 (s, 3H, CH₃), 2.3 (s, 3H,CH₃), 7.1-7.5 (m, 4H, C₆H₄F). ¹³C NMR (CDCl₃) δ: 13.0, 13.2, 115.4 (d,J=22.8 Hz), 118.1 (d, J=21.3 Hz), 123.5, 124.8 (d, J=2.6 Hz), 130.3,130.7, 140.2 (d, J=7.3 Hz), 153.0, 162.8 (d,j=246.2 Hz), 169.1 (d, J=2.6Hz). ES-MS m/z 463.1 (M+1), 485.1 (M+Na).

3-benzoyl-4,5-dimethylthiophene-2-carboxylic acid methylamide (16a). Asolution of 10 (0.40 g, 2.37 mmole) in 20 mL dry THF was cooled to −70°C. and stirred during the addition of t-butyllithium (5.21 mmole). After30 minutes of stirring benzoyl chloride (0.42 g=0.35 mL, 3 mmole) wasadded and the mixture was warmed to room temperature. Workup consistedof quenching the reaction with saturated aqueous NH₄Cl and extraction ofproduct into ethyl acetate. The extract was dried over MgSO₄, evaporatedand product purified by chromatography on silica gel eluted withhexane-ethyl acetate 1:1. Yield 0.356 g, 55% ¹H NMR(CDCl₃) δ: 1.83 (s,3H, CH₃), 2.37 (s, 3H, CH₃), 2.79 (d, 3H, NHCH₃), 6.58 (br s, 1H, NH),7.42-7.78 (m, 5H, ArH).

3-benzoyl-4,5-dimethylthiophene-2-carboxylic acid (17a). A solution of16a (0.281 g, 1.03 mmole) in methanol-water 1:1 containing 10% KOH washeated at reflux for 12 hours, neutralized and extracted with ethylacetate. The solid after evaporation was crystallized from ethanol.Yield 0.19 g, 71%.

2-Amino-3-(3-bromobenzoyl)-4,5-dihydrocyclopenta[b]thiophene (19d). Amixture of sulfur (0.176 g, 5.5 mg-at), 3-bromobenzoylacetonitrile (1.35g, 5.5 mmole) and cyclopentanone (0.463 g=0.482 mL, 5.5 mmole) in 4 mlanhydrous ethanol was heated at 50° C. in a teflon-capped pressure tubefor 4 hours. Cooling overnight deposited crystalline product, which wasfiltered off, washed with a little cold methanol and dried TLC showedthe material was pure; yield 1.2 g, 62% ¹H NMR (CDCl₃) δ: 2.16 (m, 4H,H-4 and H-6), 2.65 (m, 2H, H-5), 7.07 (br s, 2H, NH₂), 7.3-7.6 (m, 4H,C₆H₄Br).

2-Amino-3-(4-phenylbenzoyl)-4,5,6,7-tetrahydrobenzo[b]thiophene (201). Amixture of 4-phenylbenzoylacetonitrile (4.42 g, 0.02 mole),cyclohexanone (1.96 g=2.1 mL, 0.02 mole), β-alanine (0.18 g, 0.002mole), glacial acetic acid (2 mL and toluene (100 mL was heated atreflux in a flask fitted with a Deantark trap and condenser. After 18hours TLC (hexane:ethyl acetate 3:1) showed complete conversion of thenitrile, Rf 0.48, to the olefin, Rf 0.67. The residue after evaporationwas taken up in ethyl acetate washed twice with 50 mL water, dried overMgSO₄ and evaporated to a glass. Weight 4.6 g, 76%. Sulfur (0.673 g,0.021 mole) was suspended in a solution of the olefin in 50 mL anhydrousethanol, diethylamine (1 mL) was added and the dark solution was stirredat room temperature until the sulfur had disappeared. Product thatcrystallized out on cooling in an ice bath was filtered off, washed witha little methanol and dried. TLC (hexane:ethyl acetate 1:3) showed onlyproduct, Rf 0.50. Yield 4.5 g, 67% based on starting nitrite. ¹H NMR, δ:1.57 (m, 2H, cyclohexyl), 1.81 (m, 2H, cyclohexyl), 1.97 (q, 2H,cyclohexyl), 2.59 (q, 2H, cyclohexyl), 6.75 (br s, 2H, NH₂) 7.45-7.75(m, 9H, biphenyl).

Assay of AE Activity

The assay of AE activity consisted of three phases: formation of the¹²⁵I-ABA-A₁AR-G protein ternary complex; binding of the AE to theallosteric site, and dissociation of the complex by adding a combinationof an A₁AR antagonist (100 μM cyclopentyltheophylline) and 50 PM GTPγS.This procedure detects only AE activity since phase 3 (dissociation) isaffected only by the allosteric activity of the test compound and is notaffected by competitive antagonist activity. GTPγS is added toaccelerate the dissociation process. It was discovered that adding theguanine nucleotide did not interfere with AE activity, but reduced thetime needed to accurate measure AE activity during phase 3 from hours tominutes. The assay employed membranes from CHO-K1 cells stablyexpressing the hA₁AR. For agonist binding to equilibrium, the incubationmixture consisted of 10 mM HEPES, pH 7.2, containing 0.5 mM MgCl₂, 1U/mL adenosine deaminase, 0.5 nM ¹²⁵I-ABA and 10 μg of membrane proteinin a final volume of 100 μL. This phase of the assay was allowed toproceed to equilibrium (>90 minutes) at room temperature. At that pointphase 2 was initiated by adding 50 μL of a 0.3 mM solution (0.1 mMfinal) of a candidate AE or DMSO vehicle to rapidly occupy theallosteric site. Stock solutions of AEs (10 mM) were prepared in DMSO.Five minutes later phase 3 was initiated by the addition of 50 μL of asolution containing 400 μM 8-cyclopentyltheophylline and 200 μM GTPγS.Ten minutes later, residual radioligand bound to Aireceptors was trappedon Whatman GF/C glass fiber filters using a Brandel cell harvester,washed 3 times over a 20 second interval and counted in a γ-counter. Thepercentage of specifically bound agonist remaining after 10 minutes ofdissociation was used to calculate the “score” of AE activity:

 AE activity score=100×(B−B_(o))/(B_(eq−B) _(o))

Where B=residual binding (cpm) bound at the end of 10 minutes ofdissociation in the presence of an AE, B_(o)=residual binding (cpm) atthe end of 10 minutes of dissociation in the absence of an AE, andB_(eq)=cpm bound at the end of phase 2. A compound with no AE activityhas a score of 0 in this assay. An AE which completely arrests agonistdissociation will have a score of 100.

Results

The 3-aroyl moieties contributed importantly to AE activity. None of thecycloalkylthiophenes having a 3-carboxyethyl substituent, namely, 19a,20a and 21a, was active. An unsubstituted benzoyl group supported a lowlevel of AE activity, and both 3- and 4-fluorobenzoyl groups generallydid likewise. Other benzoyl substituents increased AE activity, the rankorder for all substituents being H=F<<Cl<Br<1=Ph=cHex. Both the 1- andthe 2-isomers of 3-naphthoylthiophenes had substantial AE activity. QSARanalysis¹⁵ showed that neither of the electronic parameters, σ_(m) orσ_(p), of the 3-phenyl substituent accounted for differences in AEactivity (r² for the regressions of AE data on either Hammett parameterwere <0.1 and were not significant; data not shown). However, thehydrophobic and steric parameters, π and molar refractivity,respectively, better accounted for the effect of the 3-aroylsubstituents on AE activity that the analysis could not distinguishbetween hydrophobicity and steric bulk is not surprising, since thosesubstituent parameters tend to be covariant. For the substituent groupsstudied here; r² was 0.83 for the regression of π on molar refractivity.Although most of the 3-aroyl substituents were planar, thiophenes having4phenylphenyl (19i, 20k, 21m) or 4-cyclohexylphenyl (201, 21n)substituents had excellent activity.

Table 1 lists the chemical characteristics of the novel compounds. Table2 shows the A₁AR antagonistic activity of a subset of AEs, based ontheir ability to compete with the equilibrium binding of [³H]CPX.Several of the candidate AEs had substantial antagonistic activity.However, the AE and antagonist activities are unrelated (r²=0.057,n=28). Several compounds including 21h,l had very high AE activity butwere nearly devoid of antagonist activity at 100 μM.

None of the compounds exerted AE activity at either the hA_(2A)AR or thehA₃AR. Since N⁶-substituted adenosines are agonists at both the A₁AR andA₃AR, assigning a biological response to one or the other receptor onthe basis of an agonist activity profile may give ambiguous results.Potentiation by an allosteric enhancer could be an additional criterionfor deciding that the A₁AR rather than the A₃AR initiates a response.

TABLE 1 Characteristics of Aminothiophenes No R₃—R₄—R₅ Yield %Purification Mp ° C. Formula anal  3a Ph, H, H 82 E 147 C₁₁H₉NOS C, H, N 3b 3-FPh, H, H 47 E 155 C₁₁H₈FNOS C, H, N  3c 3-CIPH, H, H 53 E 146C₁₁H₈CINOS C, H, N  3d 3-BrPH, H, H 56 E 135 C₁₁H₈BrNOS C, H, N  3e4-FPh, H, H 69 E 143 C₁₁H₈FNOS C, H, N  3f 4-CIPh, H, H 47 E 171C₁₁H₈CINOS C, H, N  3g 4-BrPh, H, H 41 E 153 C₁₁H₈BrNOS C, H, N  3h3,4-CI₂Ph, H, H 71 E 139 C₁₁H₇C₁₂NOS C, H, N  3i 2-Naph, H, H 63 E 145C₁₅H₁₁NOS C, H, N 13a Ph, Me, Me 100 E 130 C₁₃H₁₃NOS C, H, N 13b 3-FPh,Me, Me 54 E 106 C₁₃H₁₂FNOS C, H, N 13c 3-CIPh, Me, Me 38 E 115C₁₃H₁₂CINOS C, H, N 13d 3-BrPh, Me, Me 94 E 129 C₁₃H₁₂BrNOS C, H, N 13e3-CH₃Ph, Me, Me 63 E 119 C₁₄H₁₅NOS C, H, N 13g 3-PhPh, Me, Me 93 E 162C₁₉H₁₇NOS C, H, N 13h Mesityl, Me, Me 84 E 157 C₁₆H₁₉NOS C, H, N 17a Ph,2-COOH 71 E 213 C₁₃H₁₃OS C, H, N 17b 3-CF₃Ph, 2-COOH 90 E 188C₁₅H₁₁F₃O₃S C, H, N 17c 4-PhPh, 2-COOH 86 E 219 C₂₀H₁₆O₃S C, H, N 19aCO₂Et, —(CH₂)₃— 88 E 96 C₁₀H₁₃NO₂S C, H, N 19b Ph, —(CH₂)₃— 72 E 156C₁₄H₁₃NOS C, H, N 19C 3CIPh, —(CH₂)₃— 59 E 185 C₁₄H₁₂CINOS C, H, N 19d3BrPh, —(CH₂)₃— 52 E 202 C₁₄H₁₂BrNOS C, H, N 19e 4F-Ph, —(CH₂)₃— 74 E152 C₁₄H₁₂FNOS C, H, N 19f 4-CIPh, —(CH₂)₃— 72 E 125 C₁₄H₁₂CINOS C, H, N19g 4BrPh, —(CH₂)₃— 61 E 163 C₁₄H₁₂BrNOS C, H, N 19h 4PhPh, —(CH₂)₃— 31E 137 C₂₀H₁₇NOS C, H, N 19i 2-Naph, —(CH₂)₃— 55 E 155 C₁₅H₁₁NOS C, H, N20a CO₂Et, —(CH₂)₄— 73 E 96 C₁₁H₁₅NO₂S C, H, N 20b Ph, —(CH₂)₄— 64 H 143C₁₅H₁₅NOS C, H, N 20c 3-FPh, —(CH₂)₄— 52 H 113 C₁₅H₁₄FNOS C, H, N 20d3-CIPh, —(CH₂)₄— 22 H 132 C₁₅H₁₄CINOS C, H, N 20e 3-BrPH, —(CH₂)₄— 37 H120 C₁₅H₁₄BrNOS C, H, N 20f 4-FPh, —(CH₂)₄— 84 H 128 C₁₅H₁₄FNOS C, H, N20g 4-CIPh, —(CH₂)₄— 86 E 138 C₁₅H₁₄CINOS C, H, N 20h 4-BrPh, —(CH₂)₄—36 E 130 C₁₅H₁₄BrNOS C, H, N 20i 4-IPh, —(CH₂)₄— 12 E 128 C₁₅H₁₄INOS C,H, N 20j 4-CH₃Ph, —(CH₂)₄— 79 E 139 C₁₆H₁₇NOS C, H, N 20k 4-CNPh,—(CH₂)₄— 32 E 209 C₁₆H₁₄N₂OS C, H, N 20l 4-PhPh, —(CH₂)₄— 14 E 110C₂₁H₁₉NOS C, H, N 20m 4-cHexPh, —(CH₂)₄— 23 E 115 C₂₁H₂₅NOS C, H, N 20n2-Nap, —(CH₂)₄— 18 H 105 C₁₉H₁₇NOS C, H, N 21a CO₂Et, —(CH₂)₅— 75 E 117C₁₂H₁₇NO₂S C, H, N 21b Ph, —(CH₂)₅— 44 E 94 C₁₆H₁₇NOS C, H, N 21c 3CIPh,—(CH₂)₅— 21 E 75 C₁₆H₁₆CINOS C, H, N 21d 3-BrPh, —(CH₂)₅— 28 E 81C₁₆H₁₆BrNOS C, H, N 21e 3-Iph, —(CH₂)₅— 38 E 100 C₁₆H₁₆INOS C, H, N 21f4FPh, —(CH₂)₅— 28 H 79 C₁₆H₁₆FNOS C, H, N 21g 4CIPh, —(CH₂)₅— 33 H 98C₁₆H₁₆CINOS C, H, N 21h 4BrPh, —(CH₂)₅— 26 E 154 C₁₆H₁₆BrNOS C, H, N 21i4-IPh, —(CH₂)₅— 64 E 177 C₁₆H₁₆INOS C, H, N 21j 3-CH₃OPH, —(CH₂)₅— 30 E72 C₁₇H₁₉NO₂S C, H, N 21k 4-CH₃OPh, —(CH₂)₅— 84 E 125 C₁₇H₁₉NO₂S C, H, N21l 4-PhPh, —(CH₂)₅— 42 E 56 C₂₁H₂₁NOS C, H, N 21m 4-cHxPh, —(CH₂)₅— 31E 132 C₂₁H₂₇NOS C, H, N 21n 1-Naph, —(CH₂)₅— 20 E 93 C₁₉H₁₉NOS C, H, N21o 2-Naph, —(CH₂)₅— 34 E 121 C₁₉H₁₉NOS C, H, N

TABLE 2 Summary of Allosteric Enhancer Activity AE Score, Antagonist No%^(a) activity, %^(b)  3a  0.2 ± 0.01 24  3b 0  3c 0  3d  0.2 ± 0.03  3e0  3f 0  3g 0.8 ± 0.3  3h 1.7 ± 0.8  3i 3.2 ± 1.8 13a 9 13b 0 13c 14 ±1  13d 16 ± 4  13e  18 ± 0.5 13f  19 ± 2.9 42 13g  12 ± 1.9 13h 0 17a 017b 0 17c 0 19a 0.4 ± 0.1 72 19b  20 ± 3.6 63 19c 16 ± 2  19d 13 ± 3  7619e 68 ± 1  36 19f 22 ± 4  40 19g 70 ± 7  19h 62 ± 5  12 19i 38 ± 3  19j31 ± 4  23 20a 3.5 ± 1.8 57 20b 19 ± 5  35 20c 22 ± 5  40 20d 70 ± 9  220e 49 ± 1  20f 17 ± 3  51 20g 68 ± 10 9 20h 83 ± 5  56 20i 86 ± 13 3120j 19 ± 7  41 20k  19 ± 0.9 19 20l 33 ± 2  20m 99 ± 6  45 20n 86 ± 1 21a  0.3 ± 0.01 41 21b  13 ± 3.8 0 21c 65 ± 8  9 21d  78 ± 2.6 12 21e 86± 9  21f  22 ± 2.7 21g  65 ± 6.7 21h  86 ± 3.8 12 21i  96 ± 4.7 11 21j 99 ± 3.1 19 21k 85 ± 10 21l  88 ± 4.6 21m 77 ± 11 21n  81 ± 7.2 21o  75± 9.6 ^(a)See the text for a description of “score.” Mean ± SEM (N = 3).^(b)% inhibition of specific equilibrium binding of[³H]8-cyclopentyl-1,3-dipropylxanthine, N = 3.

Many improvements, modifications, and additions will be apparent to theskilled artisan without departing from the spirit and scope of thepresent invention as described herein and defined in the followingclaims.

We claim:
 1. A compound of the formula (I):

wherein: R₃ is a cycloalkylphenyl; and R₄ and R₅ are taken together toform a ring having 5 to 10 carbon atoms.
 2. The compound of claim 1wherein said cycloalkylphenyl is cyclohexylphenyl.
 3. A compound of theformula (I):

wherein: R₃ is selected from the group consisting of 1-napthyl,2-napthyl and cycloalkylphenyl; and R₄ and R₅ are taken together to forma ring having 5 to 10 carbon atoms; wherein said 1-napthyl and 2-napthylare substituted with one or more (C₁-C₆)alkyl groups, (C₂-C₆)alkenylgroups, (C₁-C₆)alkanoyl groups, (C₁-C₆)alkanoyloxy groups, (C₃-C₆)cycloalkyl groups, (C₃-C₆) cycloalkenyl groups, halo (C₁-C₆)alkylgroups, (C₁-C₆)alkoxy groups, (C₁-C₆)alkoxycarbonyl groups,(C₃-C₆)cycloalkyl(C₁-C₆)alkyl groups, (C₂-C₆)alkynyl groups or halogens.4. The compound of claim 1 wherein said ring has 5 carbon atoms.
 5. Amethod for enhancing adenosine A₁ receptors in a mammal, including ahuman, by administering to said mammal an effective amount of a compoundof formula (I):

wherein: R₃ is selected from the group consisting of 1-napthyl,2-napthyl and cycloalkylphenyl; and R₄ and R₅ are taken together to forma ring having 5 to 10 ring atoms.
 6. The method of claim 5 wherein saidcycloalkylbenzoyl is cyclohexylphenyl.
 7. The method of claim 5 whereinsaid 1-napthyl and 2-napthyl are substituted.
 8. The method of claim 7wherein said 1-napthyl and 2-napthyl are substituted with one or more(C₁-C₆)alkyl groups, (C₂-C₆)alkenyl groups, (C₁-C₆)alkanoyl groups,(C₁-C₆)alkanoyloxy groups, (C₃-C₆) cycloalkyl groups, (C₃-C₆)cycloalkenyl groups, halo (C₁-C₆)alkyl groups, (C₁-C₆)alkoxy groups,(C₁-C₆)alkoxycarbonyl groups, (C₃-C₆)cycloalkyl(C₁-C₆)alkyl groups,(C₂-C₆)alkynyl groups or halogens.
 9. The method of claim 5 wherein saidring has 5 carbon atoms.
 10. A method for promoting angiogenesis in amammal, including a human, by administering to said mammal an effectiveamount of a compound of formula (I):

wherein: R₃ is selected from the group consisting of 1-napthyl,2-napthyl and cycloalkylphenyl; and R₄ and R₅ are taken together to forma ring having about 5 to about 10 ring atoms.
 11. The method of claim 10wherein said cycloalkylphenyl is cyclohexylphenyl.
 12. The method ofclaim 10 wherein said 1-napthyl and 2-napthyl are substituted.
 13. Themethod of claim 12 wherein said 1-napthyl and 2-napthyl are substitutedwith one or more (C₁-C₆)alkyl groups, (C₂-C₆)alkenyl groups,(C₁-C₆)alkanoyl groups, (C₁-C₆)alkanoyloxy groups, (C₃-C₆) cycloalkylgroups, (C₃-C₆) cycloalkenyl groups, halo (C₁-C₆)alkyl groups,(C₁-C₆)alkoxy groups, (C₁-C₆)alkoxycarbonyl groups,(C₃-C₆)cycloalkyl(C₁-C₆)alkyl groups, (C₂-C₆)alkynyl groups or halogens.14. The method of claim 10 wherein said ring has 5 carbon atoms.
 15. Amethod of treating ischemic disease in a mammal, including a human, byadministering to said mammal an effective amount of a compound offormula (I):

wherein: R₃ is selected from the group consisting of 1-napthyl,2-napthyl and cycloalkylphenyl; and R₄ and R₅ are taken together form aring having about 5 to about 10 ring atoms.
 16. The method of claim 15wherein said cycloalkylphenyl is cyclohexylphenyl.
 17. The method ofclaim 15 wherein said 1-napthyl and 2-napthyl are substituted.
 18. Themethod of claim 17 wherein said 1-napthyl and 2-napthyl are substitutedwith one or more (C₁-C₆)alkyl groups, (C₂-C₆)alkenyl groups,(C₁-C₆)alkanoyl groups, (C₁-C₆)alkanoyloxy groups, (C₃-C₆) cycloalkylgroups, (C₃-C₆) cycloalkenyl groups, halo (C₁-C₆)alkyl groups,(C₁-C₆)alkoxy groups, (C₁-C₆)alkoxycarbonyl groups,(C₃-C₆)cycloalkyl(C₁-C₆)alkyl groups, (C₂-C₆)alkynyl groups or halogens.19. The method of claim 15 wherein said ring has 5 carbon atoms.
 20. Themethod of claim 15 wherein said ischemic disease is selected from thegroup consisting of: heart disease, stroke and peripheral vasculardisease.
 21. The method of treating cardiac arrhythmias in a mammal,including a human, by administering to said mammal an effective amountof the compound of claim
 1. 22. The method of treating chronic pain in amammal, including a human, by administering to said mammal an effectiveamount of the compound of claim
 1. 23. The method of inducing sleep in amammal, including a human, by administering to said mammal an effectiveamount of the compound of claim 1.